Figure 6.

Anomanolide C induces ferroptosis in TNBC cells in vitro and in vivo. (A) KEGG were performed to predict the potential targets. Functional clustering of gene ontology (GO) was carried out on molecular function. (B-C) Immunofluorescence analysis of Fe²⁺ levels in MDA-MB-231 and BT549 3D spheroids treated with or without AC for 24h. Quantification of immunofluorescence analysis were shown. Scale bar, 20 μm. (D-E) ROS formation in the absence or presence of AC (24 h) was observed by flow cytometry. Quantification of ROS levels and representative images are shown. (F-G) Western blotting analysis expression of FTH1, GPX4, SLC7A11 and ACSL4 treated with or without AC in MDA-MB-231 and BT549 cells. β-actin was used as a loading control. Quantification of FTH1, GPX4, SLC7A11 and ACSL4 levels and representative images are shown. (H) MDA-MB-231-luc treated nake mice tumors were observed by transmission electron microscopy. The arrow represents mitochondria. (I) Immunoblotting analysis expression of FTH1, GPX4, SLC7A11 and ACSL4 treated with AC (25, 50 mg/kg) in nake mice tumor. β-actin was used as a loading control. Quantification of FTH1, GPX4, SLC7A11 and ACSL4 levels and representative images are shown. Data are presented as the mean ± SEM. These results are consistent with those of at least three different experiments. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Statistical significance was determined relative to the appropriate AC groups.